In a study published in the journal PLOS ONE, the scientists describe how the mistakenly constructed protein mimics the action of aminoglycosides, a class of antibiotics. The newly discovered protein could serve as a model to help scientists uncover the details of these drugs’ lethal effects on bacteria – and potentially pave the way for future antibiotics.
Brookhaven scientists, who normally focus on energy-related research, did not have human health in mind when they started this project. They used E.coli bacteria to study the genes involved in building plant cell walls – research that could help scientists learn how to more efficiently convert plant matter (biomass) into biofuels. But when they turned on the expression of a particular plant gene, allowing the bacteria to make the protein, the cells immediately stopped growing.
“This protein had an extremely toxic effect on cells – all cells died within minutes of turning on the expression of this gene,” said Brookhaven biologist Paul Freimuth, who led the new research. But why would a vegetable protein cause such dramatic damage? The group discovered that the toxic factor wasn’t a plant protein at all – it was a strand of amino acids, the building blocks of protein, that made no sense.
This absurd strand had been produced by mistake when the bacteria’s ribosomes (the protein-making machinery of cells) translated the letters that make up the “out of phase” genetic code. Instead of reading the code in three-letter chunks that code for a particular amino acid, the ribosome only reads the second two letters of one chunk plus the first letter of the next triplet. This resulted in the wrong amino acids being put in place.
“It would be like reading a sentence starting in the middle of each word and joining it to the first half of the next word to produce a string of gibberish,” Freimuth said.
The gibberish protein reminded Freimuth of a class of antibiotics called aminoglycosides. These antibiotics force ribosomes to make similar “phasing” errors and other types of errors when building proteins. Result: all the ribosomes of the bacterium produce gibberish proteins.
“If a bacterial cell has 50,000 ribosomes, each producing a different aberrant protein, is the toxic effect the result of a specific aberrant protein or a combination of several? This question arose decades ago and has never been resolved,” Freimuth said.
The new research shows that a single aberrant protein may be enough for the toxic effect. That wouldn’t be too far-fetched, because absurd amino acid strands can’t fold up properly to become fully functional. Although misfolded proteins are produced in all cells by chance errors, they are usually detected and completely eliminated by “quality control” mechanisms in healthy cells. Failure of quality control systems could result in the accumulation of aberrant proteins, causing disease.
The next step was to find out if the aberrant plant protein could activate the bacterial cell’s quality control system or somehow prevent this system from working. The team found that the protein did activate the initial step of protein quality control, but the later process steps directly needed to break down aberrant proteins were blocked. They also discovered that the difference between cell life and death depended on the rate at which the aberrant protein was produced.
“When the cells contained many copies of the gene coding for the aberrant plant protein, the quality control machinery detected the protein but was unable to completely degrade it,” Freimuth said. “When we reduced the gene copy number, however, the quality control machinery was able to remove the toxic protein and the cells survived.”
The same thing happens, he noted, in cells treated with sublethal doses of aminoglycoside antibiotics. “The quality control response was strongly activated, but the cells were still able to continue growing,” he said.
These experiments indicated that the single aberrant plant protein killed cells by the same mechanism as the complex mixture of aberrant proteins induced by aminoglycoside antibiotics, although the precise mechanism of cell death is still a mystery. “The good news,” Freimuth said, “is that we now have a single protein, with a known amino acid sequence, that we can use as a model to explore this mechanism.”
Scientists already know that cells treated with antibiotics become permeable, allowing things like salts to leak in at toxic levels. One hypothesis is that misfolded proteins could form new channels in cell membranes, or block the gates of existing channels, allowing the diffusion of salts and other toxic substances across the cell membrane.
“A next step would be to determine the structures of our protein in complex with the membrane channels, in order to study how the protein could inhibit the normal function of the channels,” Freimuth said. This would help to better understand how aberrant proteins induced by aminoglycoside antibiotics kill bacterial cells – and could inform the design of new drugs to trigger the same or similar effects.
“What we discovered is far from becoming a drug,” Freimuth said. “We have identified a single protein that mimics the effect of a complex mixture of aberrant proteins produced when bacteria are treated with aminoglycosides. This gives us a way to study the mechanism that kills bacterial cells. So maybe a new family of inhibitors could be developed to do the same thing.